Knockout mice and their use

Abstract

Non-human mammals and their progenies comprising an integrin alpha10 gene, integrin alpha11 gene, or both genes are provided, wherein at least a part of the integrin alpha10 gene, the integrin alpha11 gene, or both genes, of said non-human mammal and its progeny has/have been replaced with a DNA sequence comprising at least a portion of one exon of the integrin alpha10 coding sequence, the integrin alpha11 coding sequence, or both coding sequences, linked to a selection marker sequence. Also included are methods for generating said non-human mammals with a disrupted alpha10 gene, a disrupted alpha11 gene or both genes disrupted, as well as the use of said non-human mammals.

1 . A non-human mammal and its progeny comprising an integrin alpha10 gene, wherein at least a part of an integrin alpha10 gene of said non-human mammal and its progeny has been replaced with a DNA sequence comprising at least a portion of the integrin alpha10 coding sequence linked to a selection marker sequence.  
     
     
         2 . The non-human mammal and its progeny according to  claim 1 , wherein the portion of the integrin alpha10 coding sequence is at least a portion of the integrin alpha10 intron, exon, promotor or a mixture thereof.  
     
     
         3 . The non-human mammal and its progeny according to  claim 1 , wherein the DNA sequence comprising at least a portion of one exon of the integrin alpha10 coding sequence linked to a selection marker sequence deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany, under the accession number DSM 14933.  
     
     
         4 . A non-human mammal and its progeny comprising an integrin alpha11 gene, wherein at least a part of an integrin alpha11 gene of said non-human mammal and its progeny has been replaced with a DNA sequence comprising at least a portion of the integrin alpha11 coding sequence linked to a selection marker sequence.  
     
     
         5 . The non-human mammal and its progeny according to  claim 4 , wherein the portion of the integrin alpha11 coding sequence is at least a portion of the integrin alpha11 intron, exon, promotor or a mixture thereof.  
     
     
         6 . The non-human mammal and its progeny according to  claim 4 , wherein the DNA sequence comprising at least a portion of one exon of the integrin alpha11 coding sequence linked to a selection marker sequence deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany, under the accession number DSM 14934.  
     
     
         7 . A non-human mammal and its progeny comprising an integrin alpha10 gene and an integrin alpha11 gene, wherein at least a part of an integrin alpha10 gene of said non-human mammal and its progeny has been replaced with 1) a DNA sequence comprising at least a portion of the integrin alpha10 coding sequence linked to a first marker sequence and 2) a DNA sequence comprising at least a portion of the integrin alpha11 coding sequence linked to a second selection marker sequence.  
     
     
         8 . A knockout non-human mammal and its progeny, wherein expression of the gene encoding integrin alpha10 is suppressed as compared to a wild type non-human mammal.  
     
     
         9 . The knockout non-human mammal and its progeny according to  claim 8 , wherein the suppression of the gene encoding integrin alpha10 is 100%.  
     
     
         10 . The knockout non-human mammal and its progeny according to  claim 8 , wherein the alpha10 is suppressed through insertion of a knockout construct comprising at least a portion of the integrin alpha10 coding sequence linked to a selection marker sequence.  
     
     
         11 . The non-human mammal and its progeny according to  claim 10 , wherein the portion of the integrin alpha10 coding sequence is at least a portion of the integrin alpha10 intron, exon, promotor or a mixture thereof.  
     
     
         12 . The non-human mammal and its progeny according to  claim 10 , wherein the knockout construct comprises at least a portion of one exon of the integrin alpha10 coding sequence linked to a selection marker sequence, and is deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany, under the accession number DSM 14933.  
     
     
         13 . A knockout non-human mammal and its progeny, wherein expression of the gene encoding integrin alpha11 is suppressed as compared to a wild-type non-human mammal.  
     
     
         14 . The knockout non-human mammal and its progeny according to  claim 13 , wherein the suppression of the gene encoding integrin alpha11 is 100%.  
     
     
         15 . The knockout non-human mammal and its progeny according to  claim 13 , wherein the alpha11 is suppressed through insertion of a knockout construct comprising at least a portion of the integrin alpha11 coding sequence linked to a marker sequence.  
     
     
         16 . The non-human mammal and its progeny according to  claim 15 , wherein the portion of the integrin alpha11 coding sequence is at least a portion of the integrin alpha11 intron, exon, promotor or a mixture thereof.  
     
     
         17 . The non-human mammal and its progeny according to  claim 16 , wherein the knockout construct comprises at least a portion of one exon of the integrin alpha11 coding sequence linked to a marker sequence, and is deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany, under the accession number DSM 14934.  
     
     
         18 . A knockout non-human mammal and its progeny, wherein expression of the gene encoding integrin alpha10 and the gene encoding integrin alpha11 is suppressed as compared to a wild type non-human mammal.  
     
     
         19 . The knockout non-human mammal and its progeny according to  claim 18 , wherein the suppression of the genes encoding integrin alpha10 and alpha11 is 100%.  
     
     
         20 . The knockout non-human mammal and its progeny according to  claim 18 , wherein the alpha10 is suppressed through insertion of a knockout construct comprising at least a portion of the integrin alpha10 coding sequence linked to a selection marker sequence, and wherein the alpha11 is suppressed through insertion of a knockout construct comprising at least a portion of the integrin alpha11 coding sequence linked to a second selection marker sequence.  
     
     
         21 . The non-human mammal and its progeny according to  claim 20 , wherein the portion of the integrin alpha10 coding sequence is at least a portion of the integrin alpha10 intron, exon, promotor or a mixture thereof, and wherein the portion of the integrin alpha11 coding sequence is at least a portion of the integrin alpha11 intron, exon, promotor or a mixture thereof.  
     
     
         22 . The non-human mammal and its progeny according to  claim 20 , wherein the knockout construct comprises at least a portion of one exon of the integrin alpha10 coding sequence linked to a marker sequence, and is deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany, under the accession number DSM 14933, and wherein the knockout construct comprises at least a portion of one exon of the integrin alpha11 coding sequence linked to a marker sequence, and is deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany, under the accession number DSM 14934.  
     
     
         23 . The non-human mammal and its progeny according to  claim 1 , wherein the non-human mammal and its progeny is a rodent.  
     
     
         24 . The non-human mammal and its progeny according to  claim 23 , wherein the rodent is a mouse.  
     
     
         25 . The non-human mammal and its progeny according to  claim 1 , wherein the selection marker sequence is the neomycin resistance gene.  
     
     
         26 . A method for preparing a non-human mammal and its progeny with a disrupted integrin alpha10 gene, comprising the step of replacing a portion of the integrin alpha10 gene in an embryonic stem cell by homologous recombination with a DNA sequence comprising at least a portion of one exon of the integrin alpha10 coding sequence linked to a marker sequence.  
     
     
         27 . The method according to  claim 26 , further comprising the steps of 
 a) providing a knockout construct for integrin alpha10,    b) providing an ES cell line,    c) transforming the ES cell line in b) with the construct in a),    d) selecting transformed ES cell line using a marker sequence,    e) providing a blastocyst,    f) introducing the ES cell line into the blastocyst,    g) transferring the blastocyst to a fostermother non-human mammal, and,    h) allowing an embryo to develop to a chimaeric animal to enable germline transmission of the disrupted integrin alpha10 gene.    
     
     
         28 . A vector comprising nucleic acid sequence encoding the integrin alpha10 knockout construct, wherein the sequence comprises at least a portion of one exon of the integrin alpha10 coding sequence linked to a selection marker sequence, deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany, under the accession number DSM 14933.  
     
     
         29 . An ES cell line comprising the integrin alpha10 knockout construct vector according to  claim 28 .  
     
     
         30 . A method for preparing a non-human mammal and its progeny with a disrupted integrin alpha11 gene, comprising the step of replacing a portion of the integrin alpha11 gene in an embryonic stem cell by homologous recombination with a DNA sequence comprising at least a portion of one exon of the integrin alpha11 coding sequence linked to a selection marker sequence.  
     
     
         31 . The method according to  claim 30 , further comprising the steps of 
 i) providing a knockout construct for integrin alpha11,    j) providing an ES cell line,    k) transforming the ES cell line in j) with the construct in i),    l) selecting transformed ES cell line using a marker sequence,    m) providing a blastocyst,    n) introducing the ES cell line into the blastocyst,    o) transferring the blastocyst to a fostermother non-human mammal, and,    p) allowing an embryo to develop to a chimaeric animal to enable germline transmission of the disrupted integrin alpha11 gene.    
     
     
         32 . A vector comprising nucleic acid sequence encoding the integrin alpha11 knockout construct, wherein the sequence comprises at least a portion of one exon of the integrin alpha11 coding sequence linked to a selection marker sequence, deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany, under the accession number DSM 14934.  
     
     
         33 . An ES cell line comprising the integrin alpha11 knockout construct vector according to  claim 32 .  
     
     
         34 . A method for preparing a non-human mammal and its progeny with a disrupted integrin alpha10 gene and a disrupted integrin alpha11 gene, comprising the steps of 
 q) replacing a portion of the integrin alpha10 gene in an embryonic stem cell by homologous recombination with a DNA sequence comprising at least a portion of one exon of the integrin alpha10 coding sequence linked to a selection marker sequence, and,    r) replacing a portion of the integrin alpha11 gene in an embryonic stem cell by homologous recombination with a DNA sequence comprising at least a portion of one exon of the integrin alpha11 coding sequence linked to a selection marker sequence.    
     
     
         35 . The method according to  claim 34 , further comprising the steps of 
 s) providing a knockout construct for integrin alpha10, and providing a knockout construct for integrin alpha11,    t) providing an ES cell line,    u) transforming the ES cell line in t) with the constructs in s), either at the same time or one at a time,    v) selecting transformed ES cell line using at least one marker sequence,    w) providing a blastocyst,    x) introducing the transformed and selected ES cell line into the blastocyst,    y) transferring the blastocyst to a fostermother non-human mammal, and,    z) allowing an embryo to develop to a chimaeric animal to enable germline transmission of the disrupted integrin alpha10 gene.    
     
     
         36 . A method for modulating activity in musculoskeletal and connective tissue diseases comprising utilizing the non-human mammal and its progeny according to  claim 1  as a model.  
     
     
         37 . A method for modulating activity in atherosclerosis comprising utilizing the non-human mammal and its progeny according to  claim 1  as a model.  
     
     
         38 . A method for modulating activity in fibrosis comprising utilizing the non-human mammal and its progeny according to  claim 1  as a model.  
     
     
         39 . A method for modulating activity in differentiation or function of a stem cell comprising utilizing the non-human mammal and its progeny according to  claim 1  as a model.  
     
     
         40 . The method according to  claim 39 , wherein the stem cell is selected from the group consisting of a mesenchymal stem cell, haematopoetic stem cell, and epithelial stem cell.  
     
     
         41 . A method for modulating activity in bone fracture healing comprising utilizing the non-human mammal and its progeny according to  claim 1  as a model.  
     
     
         42 . A method for modulating activity in inflammatory diseases comprising utilizing the non-human mammal and its progeny according to  claim 1  as a model.  
     
     
         43 . The method according to  claim 42 , wherein the inflammatory disease is rheumatoid arthritis or meningitis.  
     
     
         44 . A method for modulating activity of alpha10 and/or alpha11 comprising utilizing the non-human mammal and its progeny according to  claim 1  as a gene therapeutic model.  
     
     
         45 . A method for modulating activity in heart valve diseases comprising utilizing the non-human mammal and its progeny according to  claim 1  as a model.  
     
     
         46 . The method according to  claim 36 , wherein modulating activity is preventing, inhibiting, alleviating or reversing activity in said diseases.  
     
     
         47 . The method according to  claim 41 , wherein modulating activity is stimulating, preventing, inhibiting, alleviating or reversing activity in bone fracture healing.  
     
     
         48 . A method for the generation of antibodies showing reactivity with the integrin alpha10-beta1, alpha11-beta1 or parts thereof comprising utilizing the non-human mammal and its progeny according to  claim 25 .  
     
     
         49 . The method according to  claim 48 , wherein the antibodies are polyclonal antibodies.  
     
     
         50 . The method according to  claim 48 , wherein the antibodies are monoclonal antibodies.  
     
     
         51 . The method according to  claim 48 , wherein the integrin alpha10-beta1, alpha11-beta1 or parts thereof is human integin alpha10-beta1, alpha11-beta1 or parts thereof.  
     
     
         52 . A method for screening agents for effects in musculoscleletal diseases, comprising the steps of 
 i) providing a knockout mouse according to  claim 1 ,    ii) administering a test agent to said knockout mouse,    iii) determining the effect of said test agent on morphology, histology, synthesis and degradation of extracellular matrix molecules, and synthesis of integrins, and    iv) correlating the effect of said test agent in iii) above with musculoskeletal and connective tissue diseases.    
     
     
         53 . A method for screening agents for effects in atherosclerosis, comprising the steps of 
 i) providing a knockout mouse according  claim 1 ,    ii) administering a test agent to said knockout mouse,    iii) determining the effect of said test agent on morphology, histology, synthesis and degradation of extracellular matrix molecules, and synthesis of integrins, and    iv) correlating the effect of said test agent in iii) above with atherosclerosis.    
     
     
         54 . A method for screening agents for effects in fibrosis, comprising the steps of 
 i) providing a knockout mouse according to  claim 1 ,    ii) administering a test agent to said knockout mouse,    iii) determining the effect of said test agent on morphology, histology, synthesis and degradation of extracellular matrix molecules, and synthesis of integrins, and    iv) correlating the effect of said test agent in iii) above with fibrosis.    
     
     
         55 . A method for screening agents for effects in differentiation of stem cells, comprising the steps of 
 i) providing a knockout mouse according to  claim 1 ,    ii) administering a test agent to said knockout mouse,    iii) determining the effect of said test agent on morphology, histology, synthesis and degradation of extracellular matrix molecules, and synthesis of integrins, and    iv) correlating the effect of said test agent in iii) above with differentiation of stem cells.    
     
     
         56 . A method for screening agents for effects in fracture healing, comprising the steps of 
 i) providing a knockout mouse according to  claim 1 ,    ii) administering a test agent to said knockout mouse,    iii) determining the effect of said test agent on morphology, histology, synthesis and degradation of extracellular matrix molecules, and synthesis of integrins, and    iv) correlating the effect of said test agent in iii) above with fracture healing.    
     
     
         57 . A method for screening agents for effects in inflammatory diseases, comprising the steps of 
 i) providing a knockout mouse according to  claim 1 ,    ii) administering a test agent to said knockout mouse,    iii) determining the effect of said test agent on morphology, histology, synthesis and degradation of extracellular matrix molecules, and synthesis of integrins, and    iv) correlating the effect of said test agent in iii) above with inflammatory diseases.    
     
     
         58 . A method for screening agents for effects in gene therapy of alpha 10 and/or alpha 11, comprising the steps of 
 i) providing a knockout mouse according to  claim 1 ,    ii) administering a test agent to said knockout mouse,    iii) determining the effect of said test agent on morphology, histology, synthesis and degradation of extracellular matrix molecules, and synthesis of integrins, and    iv) correlating the effect of said test agent in iii) above with expression of alpha 10 and/or alpha 11.    
     
     
         59 . The method according to  claim 58 , wherein the test agent is selected from the group consisting of deoxynucleic acid, ribonucleic acid, PNA, and mixtures thereof.  
     
     
         60 . A method for generating antibody-producing hybridomas reactive to integrin alpha10-beta1 or alpha11-beta1 or parts thereof, comprising the steps of 
 i) immunizing a mouse with integrin alpha10-beta1 or parts thereof, or integrin alpha10-beta1 or parts thereof,    ii) boosting the immunized mouse in i) above with the immunized antigen,    iii) sacrifying the immunized mouse,    iv) preparing single cell suspension cells from the sacrificed mouse in iii),    vii) fusing the single cell suspension in iv) above with a tumour cell line to generate hybridoma cells,    viii) screening the hybridoma cells for reactivity to the immunized antigen.    
     
     
         61 . The method in  claim 60 , further comprising the steps of limiting dilution of screened, fused hybridoma cells in vi) above, thereby generating monoclonal hybridoma cells.